Host cells with artificial endosymbionts

ABSTRACT

The present invention is directed generally to eukaryotic host cells comprising artificial endosymbionts and methods of introducing artificial endosymbionts into eukaryotic host cells. The invention provides artificial endosymbionts that introduce a phenotype to host cells that is maintained in daughter cells. The invention additionally provides eukaryotic host cells containing magnetotactic bacteria.

FIELD OF THE INVENTION

The present invention relates generally to the field of endosymbiosis,artificial endosymbionts, and magnetotactic bacteria. In particular, theinvention provides artificial endosymbionts including magnetotacticbacteria, eukaryotic host cells for those artificial endosymbionts, andmethods of introducing the artificial endosymbionts into the cytoplasmof host cells.

BACKGROUND OF THE INVENTION

Mitochondria, chloroplast and other membrane bound organelles addheritable functionalities, such as photosynthesis, to eukaryotic cells.Such organelles (identified by their vestigial circular DNA) arebelieved to be endosymbiotically derived.

Bacteria exist with a wide range of functionalities not present invarious eukaryotic cells. For example, in 1975 Blakemore identifiedmagnetotactic bacteria (MTB) that orient and swim along a geomagneticfield. (Blakemore, R. Magnetotactic bacteria. Science 24: 377-379 (1975)(which is incorporated by reference in its entirety for all purposes)).These magnetotactic bacteria produce magnetic structures calledmagnetosomes that are composed of magnetite (Fe₃O₄) or greigite (Fe₃S₄)enclosed by a lipid membrane. (Id). A large number of MTB species havebeen identified since their initial discovery. (Id.).

Magnetotactic bacteria have been used to selectively bind to andseparate substances. (U.S. Pat. No. 4,677,067 (which is incorporated byreference in its entirety for all purposes)). Additionally, attemptshave been made to add magnetic functionality to cells through externaltags. (Swiston, A. J., Cheng, C., Soong, H. U., Irvine, D. J., Cohen, R.J., Rubner, M. F. Surface Functionalization of Living Cells withMultilayer Patches. Nano Lett. 8(12): 4446-53 (2008) (which isincorporated by reference in its entirety for all purposes)). Bacterialmagnetite has also been introduced into red blood cells by cell fusion(Matsunaga, T., Kamiya, S., (1988), In: Atsumi, K., Kotani, M., Ueno,S., Katila T., Williamsen, S. J. (eds) 6th International Conference onBiomagnetisms (1987). Tokyo Denki University Press, Tokyo, pp. 50-51(which is incorporated by reference in its entirety for all purposes)),and MTB have been introduced into granulocytes and monocytes byphagocytosis. (Matsunaga, T., Hashimoto, K., Nakamura, N., Nakamura, K.,Hashimoto, S. Phagocytosis of bacterial magnetite by leucocytes. AppliedMicrobiology and Biotechnology 31(4): 401-405 (1989) (which isincorporated by reference in its entirety for all purposes)). However,none of these alterations are heritable to daughter cells.

It is an object of the present invention to provide eukaryotic hostcells containing artificial endosymbionts that are heritable to daughtercells. It is also an object of the present invention to provide methodsof introducing artificial endosymbionts into the cytosol of eukaryotichost cells. It is another object of the present invention to provideeukaryotic host cells containing artificial endosymbionts that give thehost cell a heritable magnetic phenotype.

SUMMARY OF THE INVENTION

The present invention relates to eukaryotic host cells comprisingartificial endosymbionts and methods of introducing artificialendosymbionts into eukaryotic host cells. In one embodiment, theartificial endosymbionts are heritable to daughter cells. In anotherembodiment, the artificial endosymbiont is a magnetotactic bacterium.The artificial endosymbiont of the invention may be modified bydeleting, adding, and/or mutating at least one gene whereby theartificial endosymbiont acquires a trait useful for endosymbiosis orbiotrophy. The genes to be mutated, added, and/or deleted in theartificial endosymbiont may be genes encoding components of theflagellar assembly and genes encoding enzymes for synthesizing essentialmacromolecules, such as amino acids, nucleotides, vitamins, andco-factors. In certain embodiments, the MTB may further be modified toexpress an antibiotic resistance gene or other selectable marker.

In some embodiments the host cells of the invention are mammalian, suchas mouse, rat, rabbit, hamster, human, porcine, bovine, or canine. Inanother embodiment the artificial endosymbiont is transmitted from thehost cell to daughter progeny host cells. In another embodiment, themethod further comprises deleting, inserting, and/or mutating at leastone gene from the eukaryotic host cell.

The artificial endosymbionts of the invention can be introduced intoeukaryotic host cells by a number of methods known to those of skill inthe art including, but not limited to, microinjection, naturalphagocytosis induced phagocytosis, macropinocytosis, liposome fusion,erythrocyte ghost fusion, or electroporation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows, positive contrast generated with a T₁ pulse sequence overa log scale concentration up to ˜10⁸ MTB/mL for gfp⁺AMB suspended inagar plugs using a 1.5 T instrument to optimize and characterize theimaging properties.

FIG. 2 shows a blastula stage mouse embryo that has had one of its twocells at the 2-cell embryo stage microinjected with gfp⁺AMB. The embryois imaged with Leica SP2 AOBS spectral confocal inverted microscopesurrounded by an environmental control chamber for live-cell imagingwith 20×, 0.7 NA objective, and optical zoom of 3×. Panel A showsdifferential interference contrast (DIC) image and Panel B shows a grayscale fluorescence capture of the same image.

FIG. 3 shows the change of total embryo GFP fluorescence of four mouseembryos over time as measured by confocal microscopy. One of the twocells from the 2-cell stage of each embryo had been microinjected withgfp⁺AMB, and the total GFP fluorescence of each embryo was measuredbeginning at the 8-cell stage, 24 hours after microinjection.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by way oaf example and not by way oflimitation. It should be noted that references to “an” or “one” or“some” embodiment(s) in this disclosure are not necessarily to the sameembodiment, and all such references mean at least one.

The present invention is directed to eukaryotic host cells containingartificial endosymbionts in the cytosol of the host cell, and methods ofintroducing artificial endosymbionts into the cytosol of the host cell.In one embodiment the artificial endosymbiont is genetically altered. Insome embodiments the artificial endosymbionts are magnetotactic bacteria(MTB).

DEFINITIONS

As used herein, the term “AMB” refers to Magnetospirillum magneticumstrain AMB-1.

As used herein, the term “artificial endosymbiont” refers to a to asingle-celled organism that is or has been introduced into the cytosolof a eukaryotic cell through human intervention, which has been or canbe transferred to daughter cells of the eukaryotic cell through at leastfive cell divisions, and which maintains sufficient copy number in thedaughter cells so that a phenotype introduced by the artificialendosymbiont is maintained in the daughter cells.

As used herein, the term “cellular life cycle” refers to series ofevents involving the growth, replication, and division of a eukaryoticcell. It is divided into five stages, known as G₀, in which the cell isquiescent, G₁ and G₂, in which the cell increases in size, S, in whichthe cell duplicates its DNA, and M, in which the cell undergoes mitosisand divides.

As used herein, the term “cytosol” refers to the portion of thecytoplasm not within membrane-bound sub-structures of the cell.

As used herein, the term “daughter cell” refers to cells that are formedby the division of a cell.

As used herein, the term “essential molecule” refers to a moleculeneeded by a host cell for growth or survival.

As used herein, the term “genetically modified” refers to altering theDNA of a cell so that a desired property or characteristic of the cellis changed.

As used herein, the term “host cell” refers to a eukaryotic cell inwhich an artificial endosymbiont can reside.

As used herein, the term “liposome mediated” refers to artificialmicroscopic vesicles consisting of an aqueous core enclosed in one ormore lipid layers, used to convey artificial endosymbionts to hostcells.

As used herein, the term “magnetosome” refers to particles of magnetite(i.e., Fe₃ O₄) or greigite (Fe₃S₄) enclosed by a sheath or membrane,either as individual particles or in chains of particles.

As used herein, the term “magnetotactic bacteria” or “MTB” refers tobacteria with genes encoding magnetosomes.

As used herein, the term “mammal” refers to warm-blooded vertebrateanimals all of which possess hair and suckle their young.

As used herein, the term “microinjection” refers to the injection ofartificial endosymbionts into host cells.

As used herein, the term “tagged artificial endosymbiont” refers toartificial endosymbionts that have a ligand on the surface of theendosymbiont.

As used herein, the term “parent cell” refers to a cell that divides toform two or more daughter cells.

As used herein, the term “receptor mediated” refers to a molecularstructure or site on the surface of a host cell that binds with anartificial endosymbiont or a tagged artificial endosymbiont followed byinternalization of the artificial endosymbiont.

Artificial Endosymbionts

Artificial endosymbionts of the invention include bacteria that arecapable of surviving in a eukaryotic cell and maintain copy number suchthat the phenotype introduced by the endosymbiont is maintained indaughter cells. In some embodiments, the artificial endosymbionts do notkill the eukaryotic host cell without further human intervention. Insome embodiments, the endosymbiont can stably maintain phenotype in theeukaryotic daughter cells through at least 3 cell divisions, or at least4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 cell divisions. In another embodiment,the endosymbiont can stably maintain phenotype in the eukaryoticdaughter cells through 3-5 divisions, or 5-10 divisions, or 10-15divisions, or 15-20 divisions.

In an embodiment of the invention, the artificial endosymbionts of theinvention are genetically modified. Methods for genetically modifyingbacteria are well known in the art. Typically, the bacteria will begenetically modified to improve their survival in eukaryotic host cells,and/or to reduce the toxicity of the endosymbiont to the eukaryotic hostcell, and/or to provide the eukaryotic host cell with a usefulphenotype. In one embodiment, the flagellar proteins of an endosymbiontare modified so that the endosymbiont no longer expresses the flagellarproteins in the eukaryotic host cell. In another embodiment, theendosymbiont is modified so that it can no longer synthesize anessential molecule that is preferably provided by the eukaryotic hostcell. In an embodiment, the endosymbiont is genetically modified so thatits cell cycle is coordinated with the cell cycle of the eukaryotic hostcell so that copy number of the endosymbiont can be maintained at asufficient level to impart the phenotype to daughter cells.

Embodiments of the invention include artificial endosymbionts that areProteobacteria. Embodiments of the invention include artificialendosymbionts that are α-Proteobacteria. In the current taxonomic schemebased on 16S rRNA, α-proteobacteria are recognized as a Class within thephylum Proteobacteria, and are subdivided into 7 main subgroups ororders (Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales,Rickettsiales, Sphingomonadales and Parvularculales). (Gupta, R. S.Phylogenomics and signature proteins for the alpha Proteobacteria andits main groups. BMC Microbiology, 7:106 (2007) (which is incorporatedby reference in its entirety for all purposes)).

A large number of α-proteobacterial genomes that cover all of the maingroups within α-proteobacteria have been sequenced, providinginformation that can be used to identify unique sets of genes orproteins that are distinctive characteristics of various highertaxonomic groups (e.g. families, orders, etc.) within α-proteobacteria.(Id. (which is incorporated by reference in its entirety for allpurposes)).

Embodiments of the invention include artificial endosymbionts that aremagnetotactic bacteria (“MTB”). A large number of MTB species are knownto those of ordinary skill in the art since their initial discovery in1975 by Blakemore (Blakemore, R. Magnetotactic bacteria. Science 24:377-379 (1975) (which is incorporated by reference in its entirety forall purposes)) and represent a group of microbes (Faivre, D. & Schüler,D. Magnetotactic bacteria and magnetosomes. Chemistry Reviews 108:4875-4898 (2008) (which is incorporated by reference in its entirety forall purposes)). MTB have been identified in different subgroups of theProteobacteria and the Nitrospira phylum with most of the phylotypesgrouping in α-Proteobacteria. Currently, culturable MTB strains assignedas α-Proteobacteria by 16S rRNA sequence similarity include the strainoriginally isolated by Blakemore in 1975, Magnetospirillummagnetotactium (formerly Aquasprillium magnetotactium), M.gryphiswaldense, M. magneticum strain AMB-1 (“AMB”), M. polymorphum,Magnetosprillum sp. MSM-4 and MSM-6, Magnetococcus marinus, marinevibrio strains MV-1 and MV-2, a marine spirillum strain MMS-1 andMagnetococcus sp. strain MC-1, as well as others. A number of MTB areavailable in pure culture, including AMB. The doubling time of AMB inpure culture is approximately eight hours and is close to that of atypical mammalian cell.

Standard MTB growth media uses succinic acid as the main carbon source,but MTB can be grown with fumarate, tartrate, malate, lactate, pyruvate,oxaloacetate, malonate, P-hydroxybutyrate and maleate as the sole carbonsource. These metabolites are present inside eukaryotic cells.Microaerophillic, facultative anaerobic, and obligate anaerobic MTBstrains have been identified. Oxygen concentrations in the cytosol ofeukaryotic cells are low due to sequestration by proteins such asmyoglobin and concentration in specific cellular locations, e.g.,mitochondria, thus the microaerophilic or facultative anaerobicenvironment necessary for MTB growth is already present in theeukaryotic host cell.

MTBs can also be classified by the magnetic particles they synthesize,either magnetite (Fe₃O₄) or greigite (Fe₃S₄). Magnetite producers aremicroaerophilic or facultative anaerobic, need some oxygen source formagnetosome synthesis, and have optimal growth temperatures nearphysiological temperature.

In some embodiments, the artificial endosymbionts of the invention aregenetically modified. Molecular biology tools have been developed forgenetic manipulations of MTB most extensively in AMB and M.gryphiswaldense strain MSR-1 (reviewed in Jogler, C. and Schtiler, D. inMagnetoreception and Magnetosomes in Bacteria, New York, Springer, 2007p 134-138 (which is incorporated by reference in its entirety for allpurposes)). Since the genome of AMB was the first sequenced of any MTB,all MTB gene references herein refer to this genome unless otherwisespecified. The genomes of two other Magnetospirillum strains andMagnetococcus sp. strain MC-1 have also been recently sequenced. Genesfrom these strains or other MTB strains, presently culturable orunculturable, sequenced or unsequenced, know or unknown, can be used inthe present invention.

The genes responsible for magnetosome formation in MTB cluster ingenomic islands, known as the magnetosome island (MAI). In M.gryphiswaldense, the 130 kb MAI is generally structured into fourpolycistronic operons: the mamAB operon has 17 identified ORFs extendingover 16.4 kb; the mamGFDC operon has 4 identified ORFs is 2.1 kb and 15kb upstream ofmamAB; the mms6 operon has 6 identified ORFs is 3.6 kb and368 by upstream of the mamGFDC; the mamXY operon has 4 identified ORFsis located about 30 kb downstream of mamAB; and the monocistronic mamWgene. In the MAI the proteins: Mam W, Mgl457, Mgl458, Mgl459, Mms6,Mgl462, MamG, MamF, MamD, MamC, MamH, MamI, MamE, MamJ, MamK, MamL,MamM, MamN, MamO, MamP, MamA, MamQ, MamR, MamB, MamS, MamT, MamU andMgl505 have been identified, many of which have been given specificfunctions in magnetosome formation. Four genes outside the MAI have beenliked to magnetosome formation, mamY, mtxA, mmsF and mamX. ConservedMAI's have been found in other MTB with some differences in genomicorganization and size.

In some embodiments, genetic modifications are made to the artificialendosymbiont. Such modifications can be directed modifications, randommutagenesis, or a combination thereof. Natural endosymbionts are donorsof novel metabolic capabilities and derive nutritional requirements fromthe host.

Natural colonization of a host by the symbionts occurs in sevenstages: 1) transmission, 2) entry, 3) countering of host defense, 4)positioning, 5) providing advantage to the host, 6) surviving in hostenvironment and 7) regulation.

In some embodiments, mutual nutritional dependence (biotrophy) may beestablished between the artificial endosymbiont and the host cell. Inone embodiment, the artificial endosymbiont comprises at least onedeletion of a gene encoding an enzyme for synthesizing an essentialmolecule, wherein said essential molecule is produced by the eukaryotichost cell. An essential molecule can include, but is not limited to, anamino acid, a vitamin, a cofactor, and a nucleotide. For instance,biotrophy can be accomplished by knocking-out the ability of theartificial endosymbiont to make an amino acid, which will then bederived from the host. Glycine is a reasonable choice as it is highlyabundant in mammalian cells and a terminal product in bacterial aminoacid biogenesis; at least 22 other possibilities exist. The enzymeserine hydroxymethyltransferase converts serine into glycine at theterminus of the 3-phosphoglycerate biosynthetic pathway for amino acidproduction. In one embodiment, the artificial endosymbiont is an AMB inwhich the gene amb2339 (which encodes the enzyme serinehydroxymethyltransferase) is genetically modified. There are numerousmethods for mutating or knocking-out genes known to those of ordinaryskill in the art, including in vitro mutagenesis, targeted insertion ofDNA into the gene of interest by homologous recombination or deletion ofthe gene (or operon, as most of the genes in the bacteria cluster inoperons), or using endonucleases provided appropriate sites only aroundthe target are present in the genome.

In another embodiment, nutritional dependence for an artificialendosymbiont on the host cell could also be established by eliminatingthe ability of the artificial endosymbiont to synthesize variousmetabolites, cofactors, vitamins, nucleotides, or other essentialmolecules.

In some embodiments of the invention, an MTB artificial endosymbiont hasmutations and/or deletions in genes associated with mobility and/orsecretion. MTB are flagellated, and in some embodiments of the inventionthe MTB has a deletion and/or mutation in at least one gene encodingmolecular machinery associated with the flagella, such that the magneticbacterium does not produce a functional flagellum. Additionally, manyMTB secrete various compounds, such as hydroxamate and catecholsiderophores, which may be detrimental to or elicit an immune responsefrom the host. In the sequenced genome of AMB, of the 4559 ORF's, 83genes have been related to cell mobility and secretion. The flagellarassembly is known to be composed of the gene products of amb0498,amb0500, amb0501, amb0502, amb0503, amb0504, amb0505, amb0610, amb0614,amb0615, amb0616, amb0617, amb0618, amb0619, amb0628, amb1289, amb1389,amb2558, amb2559, amb2578, amb2579, amb2856, amb3493, amb3494 amb3495,amb3496, amb3498, amb3824, and amb3827. The flagella is controlled bythe chemotaxis machinery which is composed of at least the gene productsof amb0322, amb0323, amb0324, amb0325, amb0326, amb1806, amb1963,amb1966, amb2333, amb2635, amb2640, amb2648, amb2652, amb2826, amb2932,amb3002, amb3003, amb3004, amb3007, amb3102, amb3329, amb3501, amb3502,amb3654, amb3879 and amh3880.

In one embodiment, genes encoding antibiotic resistance are insertedinto the genome of the artificial endosymbiont. Host cells cultured inmedia containing the antibiotic will require the artificial endosymbiontfor survival. Neomycin resistance is conferred by either one of twoaminoglycoside phosphotransferase genes, which also provide resistanceagainst geneticin (G418), a commonly used antibiotic for eukaryotes.Hygromycin B resistance is conferred by a kinase that inactivateshygromcin B by phosphorylation. Puromycin is a commonly used antibioticfor mammalian cell culture and resistance is conferred by the pac geneencoding puromycin N-acetyl-transferase. External control of theantibiotic concentration allows intracellular regulation of the copynumber of the artificial endosymbionts. Any other system whereresistance or tolerance to an external factor is achieved by chemicalmodification of this factor can also be employed. An indirect nutritiveadvantage on host cells may also be established by using MTB artificialendosymbionts and a magnetic culture method. In this embodiment,magnetic fields are established to confer an advantage to cells with MTBartificial endosymbionts. This could be either by providing the meansfor attachment to culture matrix or the access to necessary growth ormedia factors.

In another embodiment, genetic modifications are made the MTB genome toenhance intracellular stability against the host defense mechanisms fora particular host cell type. Many eukaryote endosymbionts andendoparasites, such as the proteobacterial endosymbionts of insects suchas Buchnera, Wigglesworthia, and Wolhachia; the methanogenicendosymbionts of anaerobic ciliates; the nitrogen-fixing symbionts inthe diatom Rhopalodia; the chemosynthetic endosymbiont consortia ofgutless tubeworms (Olavius or Jnanidrillus), the cyanobacterialendosymbionts of sponges, the endosymbionts of all five extant classesof Echinodermata, the Rhizobia endosymbionts of plants, variousendosymbiotic algae, the Legionella-like X bacteria endosymbionts ofAmeoba proteus, numerous Salmonella sp., Mycobacterium tuberculosis,Legionella pneumophila, etc. reside in membrane-bound vacuoles oftentermed symbiosomes, while some species, such as Blochmannia, therickettsia, Shigella, enteroinvasive Escherichia coli, and Listeria,have the ability to inhabit the cytosol. The Dot-Icm Type IV secretorysystem is employed by many intracellular bacteria acquired byphagocytosis to evade the endocytic pathway and persist in the hostcell. This system has been well-studied in L. pneumophila and consistsof the proteins: DotA through DotP, DotU, DotV, IcmF, IcmQ through IcmT,IcmV, IcmW and IcmX. In Photorhabdus luminescens, the luminescentendosymbiont of nematodes, the genes encoding RTX-like toxins,proteases, type III secretion system and iron uptake systems were shownto support intracellular stability and replication. The gene bacA andthe regulatory system BvrRS are essential for maintenance of symbiosisbetween Rhizobia and plants as well as the survival of Brucella ahortusin mammalian cells. The PrfA regulon enables some Listeria species toescape the phagesome and inhabit the cytosol. The desired cellularlocation (e.g., symbiosome or cytosol) of the intracellular MTB willdictate which genes are required to be expressed in the MTB (eitherdirectly from the genome or through a stable vector) for survival andproliferation in the host environment. The endogenous plasmid pMGT ishighly stable in MTB and a number of other broad range vectors (thoseoflncQ, IncP, pBBR1, etc.) are capable of stable replication in MTB.

In another embodiment, the artificial endosymbiont is geneticallymodified by knocking in genes, such as bacteriostatic gene(s),siderophore gene(s), metabolic requirement gene(s), suicide gene(s),life cycle regulation gene(s), transporter gene(s), and escape from thephagosome gene(s). In another embodiment, the artificial endosymbiontsare randomly mutated and subsequently screened for enhanced integrationwithin the host cell. Random mutation can be accomplished by treatmentwith mutagenic compounds, exposure to UV-light or other methods know tothose skilled in the art.

In another embodiment, transgenetic modification(s) are made to counterhost cell defenses using genes from various parasites or endosymbionts.In another embodiment, the population of the artificial endosymbionts inthe host cell cytosol is regulated though a balance of intrinsic use ofhost mechanisms (nutrient availability, control of reproduction, etc.)and antibiotic concentration.

In another embodiment, a natural endosymbiont or an intracellularparasite is genetically modified into an artificial endosymbiont toproduce magnetosomes. Endosymbionts of insects such as Buchnera,Wigglesworthia, and Wolbachia; the methanogenic endosymbionts ofanaerobic ciliates; the nitrogen-fixing symbionts in the diatomRhopalodia; the chemosynthetic endosymbiont consortia of gutlesstubeworms (Olavius or Inanidrillus), the cyanobacterial endosymbionts ofsponges, the endosymbionts of all five extant classes of Echinodermata,the Rhizobia endosymbionts of plants, various endosymbiotic algae, theLegionella-like X bacteria endosymbionts of Ameoba proteus, numerousSalmonella sp., Mycobacterium tuberculosis, Legionella pneumophilabelong to α-proteobacteria and could be genetically engineered toproduce magnetosomes. In another embodiment, a pre-existing organellecan be genetically modified to express one or more magnetosome genes toproduce an artificial endosymbiont. For instance, mitochondria,plastids, hydrogenosomes, apicoplasts or other organelles, which harbortheir own genetic material, can be genetically altered.

In a preferred embodiment, the artificial endosymbiont is an MTB, whichmay or may not be genetically altered, that produces magnetic particlesupon culturing of the eukaryotic host cells.

Eukaryotic Host Cells

The invention provides eukaryotic host cells comprising artificialendosymbionts in the host cells and methods of introducing theartificial endosymbionts into host cells. In some embodiments the hostcells of the invention are mammalian, such as mouse, rat, rabbit,hamster, human, porcine, bovine, or canine. Mice routinely function as amodel for other mammals, most particularly for humans. (See, e.g.,Hanna, J., Wernig, M., Markoulaki, S., Sun, C., Meissner, A., Cassady,J. P., Beard, C., Brambrink, T., Wu, L., Townes, T. M., Jaenisch, R.Treatment of sickle cell anemia mouse model with iPS cells generatedfrom autologous skin. Science 318: 1920-1923 (2007); Holtzman, D. M.,Bales, K. R., Wu, S., Bhat, P., Parsadanian, M., Fagan, A., Chang, L.K., Sun, Y., Paul, S. M. Expression of human apolipoprotein E reducesamyloid-β deposition in a mouse model of Alzheimer's disease. J. Clin.Invest. 103(6): R15-R21 (1999); Warren, R. S., Yuan, H., Matli, M. R.,Gillett, N. A., Ferrara, N. Regulation by vascular endothelial growthfactor of human colon cancer tumorigenesis in a mouse model ofexperimental liver metastasis. J. Clin. Invest. 95: 1789-1797 (1995)(each of these three publications is incorporated by reference in itsentirety for all purposes)).

In some embodiments, the host cell is a human cancer cell. There aremany human cancer cell lines that are well known to those of ordinaryskill in the art, including common epithelial tumor cell lines such asCoco-2, MDA-MB231 and MCF7, non-epithelial tumor cell lines, such asHT-1080 and HL60, the NCI60-cell line panel (see, e.g., Shoemaker, R.,The NCI60 human tumour cell line anticancer drug screen. Nature ReviewsCancer 6, 813-823 (2006) (which is incorporated by reference in itsentirety for all purposes)). Additionally, those of ordinary skill inthe art are familiar with obtaining cancer cells from primary humantumors.

In other embodiments, the host cells are stem cells. Those of ordinaryskill in the art are familiar with a variety of stem cell types,including Embryonic Stem Cells, Inducible Pluripotent Stem Cells,Hematopoietic Stem Cells, Neural Stem Cells, Epidermal Neural Crest StemCells, Mammary Stem Cells, Intestinal Stem Cells, Mesenchymal stemcells, Olfactory adult stem cells, and Testicular cells.

In an embodiment, the eukaryotic host cell is a cell found in thecirculatory system of a human host. For example, red blood cells,platelets, plasma cells, T-cells, natural killer cells, or the like, andprecursor cells of the same. As a group, these cells are defined to becirculating host cells of the invention. The present invention may beused with any of these circulating cells. In an embodiment, theeukaryotic host cell is a T-cell. In another embodiment, the host cellis a B-cell. In an embodiment the eukaryotic host cell is a neutrophil.In an embodiment, the eukaryotic host cell is a megakaryocyte.

In another embodiment, at least one gene from the eukaryotic host cellis genetically altered. In some embodiments, mutual nutritionaldependence (biotrophy) may be established between the artificialendosymbiont and the host cell by genetic modification of the host cell,using the appropriate molecular biology techniques specific to thetarget host cell type known to those of ordinary skill in the art,creating host dependence on the artificial endosymbiont for someessential macromolecule thus establishing the environmental pressuresfor biotrophy. In another embodiment, nutritional dependence for anartificial endosymbiont on the host cell may be established bygenetically altering the host cell to eliminate the ability of theartificial endosymbiont to synthesize various metabolites, cofactors,vitamins, nucleotides, or other essential molecules. In suchembodiments, the essential molecule may be provided by the artificialendosymbionts. In another embodiment, the eukaryotic host cell geneencoding the enzyme serine hydroxymethyltransferase, which convertsserine into glycine at the terminus of the 3-phosphoglyceratebiosynthetic pathway for amino acid production, may be modified.

Methods of Introducing Artificial Endosymbionts to Host Cells

The artificial endosymbionts of the invention can be introduced intoeukaryotic host cells by a number of methods known to those of skill inthe art including, but not limited to, microinjection, naturalphagocytosis, induced phagocytosis, macropinocytosis, liposome fusion,erythrocyte ghost fusion, electroporation, receptor mediated methods,and the like. (See Microinjection and Organelle TransplantationTechniques, Celis et al. Eds.; Academic Press: New York, 1986 andreferences therein, (incorporated by reference in its entirety for allpurposes)).

In one embodiment, the artificial endosymbiont is introduced to the hostcell by microinjection into the cytoplasm of the host cell. A variety ofmicroinjection techniques are known to those skilled in the art.Microinjection is the most efficient of transfer techniques available(essentially 100%) and has no cell type restrictions (Id.; Xi, Z. &Dobson, S. Characterization of Wolbachia transfection efficiency byusing microinjection of embryonic cytoplasm and embryo homogenate. Appl.Environ. Microbiol. 71(6): 3199-3204 (2005); Goetz, M., Bubert, A.,Wang, G., Chico-Calero, 1., Vazquez-Boland, J. A., Beck, M., Slaghuis,J., Szalay, A. A., Goebel, W. Microinjection and growth of bacteria inthe cytosol of mammalian host cells. Proc. Natl. Acad. Sci. USA98:12221-12226 (2001) (each of these three publications is incorporatedby reference in its entirety for all purposes)).

Naturally phagocytotic cells have been show to take up bacteria,including MTB (Burdette, D. L., Seemann, J., Orth, K. Vibrio VopQinduces PI3-kinase independent autophagy and antagonizes phagocytosis.Molecular microbiology 73: 639 (2009); Wiedemann, A., Linder, S.,Grassi, G., Albert, M., Autenrieth, I., Aepfelbacher, M. Yersiniaenterocolitica invasin triggers phagocytosis via β1 integrins, CDC42Hsand WASp in macrophages. Cellular Microbiology 3: 693 (2001); Hackam, D.J., Rotstein, O. D., Schreiber, A., Zhang. W., Grinstein, S. Rho isrequired for the initiation of calcium signaling and phagocytosis by Fcγreceptors in macrophages, J. of Exp. Med. 186(6): 955-966 (1997);Matsunaga, T., Hashimoto, K., Nakamura, N., Nakamura, K., Hashimoto, S.Phagocytosis of bacterial magnetite by leucocytes. Applied Microbiologyand Biotechnology 31(4): 401-405 (1989) (each of these our publicationsis incorporated by reference in its entirety for all purposes)).

This method is scalable, but may be limited to specific cell types(e.g., macrophage). However, recent studies have shown thatnon-phagocytotic cell types can be induced to endocytose bacteria whenco-cultured with various factors: media and chemical factors, biologicfactors (e.g., baculovirus, protein factors, genetic knock-ins, etc.).(See, e.g., Salminen, M., Airenne, K. J., Rinnankoski, R., Reimari, J.,Valilehto, O., Rinne, J., Suikkanen, S., Kukkonen, S., Yla-Herttuala,S., Kulomaa, M. S., Vihinen-Ranta, M. Improvement in nuclear entry andtransgene expression of baculoviruses by disintegration of microtubulesin human hepatocytes. J. Virol. 79(5): 2720-2728 (2005); Modalsli, K.R., Mikalsen, S., Bukholm, G., Degre, M. Microinjection of HEp-2 cellswith coxsackie B1 virus RNA enhances invasiveness of Shigella flexnerionly after prestimulation with UV-inactivated virus. APMIS 101: 602-606(1993); Hayward, R. D. & Koronakis, V. Direct nucleation and bundling ofactin by the SipC protein of invasive Salmonella. The EMBO Journal 18:4926-4934 (1999); Yoshida, S., Katayama, E., Kuwae, A., Mimuro, H.,Suzuki, T., Sasakawa, C. Shigella deliver an effector protein to triggerhost microtubule destabilization, which promotes Rac 1 activity andefficient bacterial internalization. The EMBO Journal 21: 2923-2935(2002); Bigildeev et al. J. Exp Hematol., 39: 187 (2011); Finlay, B. B.& Falkow, S. Common themes in microbial pathogenicity revisited.Microbiol. and Mol. Biol. Rev. 61: 136-169 (1997) (each of these sixpublications is incorporated by reference in its entirety for allpurposes).

The related process, macropinocytosis or “cell drinking,” is a methodnumerous bacteria and viruses employ for intracellular entry (Zhang(2004) In: Molecular Imaging and Contrast Agent Database (MICAD)[database online]; Bethesda (Md.): National Library of Medicine (US),NCBI; 2004-2011 (each of these two publications is incorporated byreference in its entirety for all purposes)). Various protocols existwhich can be employed to induce cells to take up bacteria.

Several agents, such as nucleic acids, proteins, drugs and organelleshave been encapsulated in liposomes and delivered to cells (Ben-Haim,N., Broz, P., Marsch, S., Meier, W., Hunziker, P. Cell-specificintegration of artificial organelles based on functionalized polymervesicles. Nano Lett. 8(5): 1368-1373 (2008); Lian, W., Chang, C., Chen,Y., Dao, R., Luo, Y., Chien, J., Hsieh, S., Lin, C. Intracellulardelivery can be achieved by bombarding cells or tissues with acceleratedmolecules or bacteria without the need for carrier particles.Experimental Cell Research 313(1): 53-64 (2007); Heng, B. C. & Cao, T.Immunoliposome-mediated delivery of neomycin phosphotransferase for thelineage-specific selection of differentiated/committed stem cellprogenies: Potential advantages over transfection with marker genes,fluorescence-activated and magnetic affinity cell-sorting. MedHypotheses 65(2): 334-336 (2005); Potrykus (1990) Ciba Found Symp, Vol.1 54: 198 (each of these four publications is incorporated by referencein its entirety for all purposes)). This method is inexpensive,relatively simple and scalable. Additionally, liposome uptake can beenhanced by manipulation of incubation conditions, variation of liposomecharge, receptor mediation, and magnetic enhancement. (See, e.g.,

Pan et al. Int. J. Pharm. 358: 263 (2008); Sarbolouki, M. N. & Toliat,T. Storage stability of stabilized MLV and REV liposomes containingsodium methotrexate (acqueous & lyophilized). J. Pharm. Sci. Techno.,52(10): 23-27 (1998); Elorza, B., Elorza, M. A., Sainz, M. C., Chantres,J. R. Comparison of particle size and encapsulation parameters of threeliposomal preparations. J. Microencapsul. 10(2): 237-248 (1993);Mykhaylyk, O., Sánchez-Antequera, Y., Vlaskou, D., Hammerschmid, E.,Anton, M., Zelphati, O. and Plank, C. Liposomal Magnetofection. MethodsMol. Bio., 605: 487-525 (2010) (each of these four publications isincorporated by reference in its entirety for all purposes)).

Erythrocyte-mediated transfer is similar to liposome fusion and has beenshown to have high efficiency and efficacy across all cell types tested(Microinjection and Organelle Transplantation Techniques; Celis et al.Eds.; Academic Press: New York, 1986 (which is incorporated by referencein its entirety for all purposes)). Typically erythrocytes are loaded byosmotic shock methods or electroporation methods (Schoen, P., Chonn, A.,Cullis, P. R., Wilschut, J., and Schuerrer, P. Gene transfer mediated byfusion protein hemagglutinin reconstituted in cationic lipid vesicles.Gene Therapy 6: 823-832 (1999); Li, L. H., Hensen, M. L., Zhao, Y. L.,Hui, S. W. Electrofusion between heterogeneous-sized mammalian cells ina pellet: potential applications in drug delivery and hybridomaformation. Biophysical Journal 71:479-486 (1996); Carruthers, A., &Melchior, D. L. A rapid method of reconstituting human erythrocyte sugartransport proteins. Biochem. 23: 2712-2718 (1984) (each of these threepublications is incorporated by reference in its entirety for allpurposes). Alternatively, erythrocytes may be loaded indirectly byloading hematopoietic progenitors with artificial endosymbionts andinducing them to differentiate and expand into erythrocytes containingartificial endosymbionts.

Electroporation is a commonly used, inexpensive method to deliverfactors to cells. (Potrykus, I. Gene transfer methods for plants andcell cultures. Ciba Found Symp 154, 198-208; discussion 208-112 (1990);Wolbank, S. et al. Labelling of human adipose-derived stem cells fornon-invasive in vivo cell tracking. Cell Tissue Bank 8, 163-177 (2007)(each of these two publications is incorporated by reference in itsentirety for all purposes)).

In another embodiment, a eukaryotic host cell that naturally endocytosesbacteria (e.g., Chinese hamster ovary (CHO)) is used. In one embodiment,the modified artificial endosymbionts are added to the CHO culturedirectly. CHO cells are cultured by standard procedures in Ham's F-12media with 10% fetal calf serum media prior to infection with the MTB.Post infection, the media is augmented with additional iron (40 to 80μM) as either ferric malate or FeCl₃. Numerous other cell typesinternalize bacteria by endocytosis or more specifically phagocytosis;endosymbionts or parasites have their own methods for cellular entry andthese natural processes can be exploited for internalization of theartificial endosymbionts resulting in the generation of so-calledsymbiosomes. In another embodiment, symbiosomes from one cell can betransplanted to another cell type (i.e., one incapable of endocytosis ofartificial endosymbionts) using microinjection, organelletransplantation, and chimera techniques. These host cells are culturedin typical media and with the techniques for the specific cell type.

In one embodiment, the artificial endosymbiont is introduced to the hostcell by a liposome mediated process. Mitochondria and chloroplasts,which are larger than MTB, have been efficiently introduced intoeukaryotic cells when encapsulated into liposomes. (Bonnett, H. T.Planta 131, 229 (1976); Giles, K.; Vaughan, V.; Ranch, J.; Emery, J.Liposome-mediated uptake of chloroplasts by plant protoplasts. In VitroCellular & Developmental Biology—Plant 16(7) 581-584 (each of these twopublications is incorporated by reference in its entirety for allpurposes)). Numerous liposome fusion protocols and agents are availableand can be used by the skilled artisan without undue experimentation.(See, e.g., Ben-Haim, N., Broz, P., Marsch, S., Meier, W., Hunziker, P.Cell-specific integration of artificial organelles based onfunctionalized polymer vesicles. Nano Lett. 8(5): 1368-1373 (2008);Lian, W., Chang, C., Chen, Y., Dao, R., Luo, Y., Chien, J., Hsieh, S.,Lin, C. Intracellular delivery can be achieved by bombarding cells ortissues with accelerated molecules or bacteria without the need forcarrier particles. Experimental Cell Research 313(1): 53-64 (2007);Heng, B. C. & Cao, T. Immunoliposome-mediated delivery of neomycinphosphotransferase for the lineage-specific selection ofdifferentiated/committed stem cell progenies: Potential advantages overtransfection with marker genes, fluorescence-activated and magneticaffinity cell-sorting. Med Hypotheses 65(2): 334-336 (2005); Potrykus(1990) Ciba Found Symp, Vol. 1 54: 198 (each of these four publicationsis incorporated by reference in its entirety for all purposes)).

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter.

EXAMPLES Example 1

Microinjection of Gfp⁺AMB into Murine Cells

A. Construction of gfp⁺AMB.

Expression vectors for eGFP, one including a Shine-Dalgarno sequenceupstream of the gfp gene and one without a Shine Dalgarno, sequence werecloned into cryptic broad host range vector pBBR1MCS-2 (Kovach, M. E.,et al. Four new derivatives of the broad-host-range cloning vectorpBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166,175-176, (1995) (which is incorporated by reference in its entirety forall purposes)). AMB (ATCC 700264) was transformed with this construct.(Matsunaga, T. et al. Complete genome sequence of the facultativeanaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1. DNARes. 12, 157-166 (2005); Burgess J. G., et al. Evolutionaryrelationships among Magnetospirillum strains inferred from phylogeneticanalysis of 16S rDNA sequences. J. Bacteriol. 175: 6689-6694 (1993);Matsunaga T, et al. Gene transfer in magnetic bacteria: transposonmutagenesis and cloning of genomic DNA fragments required formagnetosome synthesis. J. Bacteriol. 174: 2748-2753 (1992); Kawaguchi R,et al. Phylogeny and 16s rRNA sequence of Magnetospirillum sp. AMB-1, anaerobic magnetic bacterium. Nucleic Acids Res. 20: 1140, (1992) (each ofthese four publications is incorporated by reference in its entirety forall purposes)).

Transformation was achieved by conjugation using a donor Escherichiacoli strain as described by Goulian, M. van der Woude, M. A. A simplesystem for converting lacZ to gfp reporter fusions in diverse bacteria.Gene 372, 219-226 (2006); Scheffel, A. Schüler, D. The Acidic RepetitiveDomain of the Magnetospirillum gryphiswaldense MamJ Protein DisplaysHypervariability but Is Not Required for Magnetosome Chain Assembly. JBacteriol. September; 189(17): 6437-6446 (2007) (each of these twopublications is incorporated by reference in its entirety for allpurposes). The mating reactions were cultured for 10 days under definedmicroaerophilic conditions in the absence of DAP to select for positivetransformants.

Following conjugation, gfp⁺AMB transformants with and without theShine-Dalgarno sequence successfully displayed GFP fluorescence. Thetranformants containing the Shine-Dalgarno sequence displayed higherlevels of GFP fluorescence than the transformance without this sequence.The resulting fluorescence did not leave the gfp⁺AMB cells when viewedat 100× magnification at 488 nm excitation.

The magnetic properties of the gfp⁺AMB were analyzed by MRI. The gfp⁺AMBwas suspended in agar plugs using a 1.5 T instrument to optimize andcharacterize the imaging properties. FIG. 1 shows the positive contrastgenerated with a T₁ pulse sequence over a log scale concentration up to˜10⁸ MTB/mL. Signal intensity was related to concentration.

B. Microinjection into Murine Embryonic Cells.

The gfp⁺AMB was mircoinjected into one cell of each of 170 mouse embryosat the 2-cell stage. Six concentrations over a log scale up to ˜10⁵gfp⁺AMB were injected per cell, estimated by the optical density at 565nm. Death rate of cells following microinjection was constant across thedifferent injected concentrations. Images overlaying fluorescent anddifferential interference contrast (DIC) images of cells injected withthe highest concentration (10⁵ MTB/cell) were compared. An uninjectedcontrol exhibited low levels of autofluorescence. Slices at differenthorizontal planes in 8-cell embryos at a given time point were compared.In each embryo, all four cells derived from the injected cell showedsignificant fluorescence while none of the four cells derived from theuninjected internal controls displayed significant fluorescence.

The embryos were allowed to develop for three days after the injection.In each concentration level, embryos survived for up to the full threedays developing to the 256 cell blastula stage and appeared healthyenough for implantation. Numerous cells within each blastula displayedsignificant fluorescence, demonstrating that the artificialendosymbionts were transferred to daughter cells across multiple celldivisions as the embryos comprising the eukaryotic host cells developedto the blastula stage. One such blastula is shown in FIG. 2, where PanelA shows a differential interference contrast (DIC) image of the blastulaand Panel B) shows a gray scale fluorescence capture of the same image,showing fluorescence in numerous cells throughout the blastula.

Confocal microscopy was used to quantify total expression of GFPthroughout four individual embryos by measuring total GFP fluorescencein the entire embryo over time at various points beginning at the eightcell stage of the embryo. FIG. 3 shows the change of embryo fluorescenceover time. This indicates that the copy number of artificialendosymbionts was maintained in daughter cells for at least sevengenerations, such that the fluorescent phenotype of the host cells wasmaintained as the embryo progressed from the 2-cell stage to the256-cell blastula stage.

These results demonstrate that, when delivered by microinjection,gfp⁺AMB were not immediately cleared or degraded and were not toxic tothe developing embryo over the course of the three day experiment.Microinjected embryos divided normally, suggesting that gfp⁺AMB do notdisplay pathogenic markers or secret toxic compounds. They weretransferred to daughter cells across many cell divisions, were containedin the cytoplasm, were punctate and well distributed, and maintainedcopy number within the daughter host cells, such that the fluorescentphenotype of the eukaryote host cells was maintained in daughter cellsthrough at least seven generations. These results demonstrate that AMBcan be stably maintained intracellularly and are transferred to daughtercells over at least seven cell divisions.

Example 2 Phagocytic Entry of AMB

Receptor mediated: The inlAB gene is amplified from L. monocytogenesgenomic DNA (ATCC 11911D-5) and is inserted into pBBR1MCS-5,107 thegentamicin cognate of pBBR1MCS-2 (Kovach, M. E., et al. Four newderivatives of the broad-host-range cloning vector pBBR1MCS, carryingdifferent antibiotic-resistance cassettes. Gene 166, 175-176, (1995)),and gfp+inlAB+ AMB is generated. The gfp+inlAB+ AMB is co-cultured witheukaryotic host cells, including common epithelial tumor cell linesCoco-2, MDA-MB231 and MCF7, non-epithelial tumor cell lines, such asHT-1080 and HL60, and murine stem cells. Fluorescent microscopy and FACSare used to monitor and quantify internalization and intracellularlocation.

Expression of pore-forming haemolysin (hlyA) in AMB is achieved throughamplification of hlyA from L. monocytogenes genomic DNA (ATCC 11911D-5).The amplified hlyA is inserted into pBBR1MCS-3 (the tetracycline cognateof pBBR1MCS-2) which is then used to transform gfp⁺AMB. The resultingAMB strain is exposed to murine macrophage cell line J774, capable ofspontaneous phagocytosis. Gentomycin treatment is used to eliminatebacteria not internalized and hlyA⁻AMB is used as negative control.Fluorescent microscopy is used to monitor the intracellular fate andlocalization of AMB.

If bacteria remain confined to the phagosomes, we will introduce twogenes, plcA and plcB, implicated in escape of L. monocytogenes into thecytosol. (Smith, G. A., Marquis, H., Jones, S., Johnston, N. C.,Portnoy, D. A., Goldfine, H. Infection and immunity 63: 4231 (1995);Camilli, A.; Goldfine, H.; Portnoy, D. A. The Journal of ExperimentalMedicine 173: 751 (1991) (each of these two publications is incorporatedby reference in its entirety for all purposes)). If bacteria escapesuccessfully, but fail to propagate, we will introduce hpt. (Goetz, M.,Bubert, A., Wang, G., Chico-Calero, I., Vazquez-Boland, J. A., Beck, M.;Slaghuis, J., Szalay, A. A., Goebel, W. Proc Natl Acad Sci USA 98: 12221(2001); Chico-Calero, I., Suarez, M., Gonzalez-Zorn, B., Scortti, M.,Slaghuis, J., Goebel, W., Vazquez-Boland, J. A. Proc Natl Acad Sci USA99: 431 (2002) (each of these two publications is incorporated byreference in its entirety for all purposes)). In L. monocytogenes, hptencodes the transporter responsible for uptake of glucose-6-phosphatefrom the cytosol. Other genes from L. monocytogenes have been implicatedin sustaining growth within host (glnA and gltAB and argD) and we willsystematically introduce these as needed. (Joseph, B., Przybilla, K.,Stuhler, C., Schauer, K., Slaghuis, J., Fuchs, T. M., Goebel, W. Journalof Bacteriol. 188: 556 (2006) (which is incorporated by reference in itsentirety for all purposes)).

Example 3

Regulation of AMB Growth

Regulation of AMB growth in embryonic stem cells can be regulated asfollows. Coleoptericin-A (ColA) is amplified from total Sitophilusoryzae cDNA. Expression of ColA in beetles of genus Sitophilus regulatestiters of γ-Protobacterium, which has naturally developed closesymbiotic relationship the beetles, and resides in specific cells calledbacteriocytes. (Login, F. H., Balmain, S., Vallier, A., Vincent-Monegat,C., Vigneron, A., Weiss-Gayet, M., Rochat, D., Heddi, A. Antimicrobialpeptides keep insect endosymbionts under control. Science 334(6054):362-365 (2011) (which is incorporated by reference in its entirety forall purposes)).

Murine embryonic stem cells comprising gfp+AMB are treated using aneural differentiation protocol. MTB expression levels are quantifiedusing qPCR and fluorescent microscopy. Amplified colA is then expressedin the gfp+AMB embryonic stem cells. A promoter is selected to provideoptimal ColA expression levels.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A mammalian host cell comprising at least one magnetotactic bacterium, wherein the magnetotactic bacterium comprises a fluorescent reporter.
 2. The mammalian host cell of claim 1, wherein the fluorescent reporter is a GFP.
 3. The mammalian host cell of claim 1, wherein the magnetotactic bacterium further comprising a pore-forming haemolysin encoded by a hlyA gene of L. monocytogenes.
 4. The mammalian host cell of claim 1, wherein the mammalian host cell is a circulatory cell.
 5. The mammalian host cell of claim 4, wherein the circulatory cell is a T-cell.
 6. The mammalian host cell of claim 1, wherein the mammalian cell is a human cell.
 7. The mammalian host cell of claim 1, wherein the mammalian cell is a murine cell.
 8. The mammalian host cell of claim 1, wherein the mammalian cell is a cancer cell.
 9. The mammalian host cell of claim 1, wherein the mammalian cell is a stem cell.
 10. The mammalian host cell of claim 9, wherein the stem cell is a neural stem cell.
 11. A method of detecting a mammalian cell comprising the steps of: obtaining the mammalian cell wherein the mammalian cell comprises a magnetotactic bacterium, and wherein the magnetotactic bacterium comprises a fluorescent reporter, and detecting the fluorescent reporter.
 12. The method of claim 11, wherein the fluorescent reporter is a GFP.
 13. The method of claim 11, wherein the magnetotactic bacterium further comprises a pore-forming haemolysin encoded by a hlyA gene of L. monocytogenes.
 14. The method of claim 11, wherein the mammalian cell is a circulatory cell.
 15. The method of claim 14, wherein the circulatory cell is a T-cell.
 16. The method of claim 11, wherein the mammalian cell is a human cell.
 17. The method of claim 11, wherein the mammalian cell is a murine cell.
 18. The method of claim 11, wherein the mammalian cell is a cancer cell.
 19. The method of claim 11, wherein the mammalian cell is a stem cell.
 20. The method of claim 19, wherein the stem cell is a neural stem cell. 